JP4516258B2 - Novel proteins, genes encoding the same, and uses thereof - Google Patents

Abstract

The object of the present invention is to screen and identify a novel antimicrobial protein which can inhibit the growth of plant pathogenic microorganisms at a relatively low concentration such as Pyricularia oryzae and Rhizoctonia solani causative of two major diseases causing damage to rice crops and, further, to clone the gene of this protein. According to the present invention, an antimicrobial protein which can be obtained from a fraction of an aqueous extract of Lyophyllum shimeji precipitated by the ammonium sulfate precipitation method, has an antimicrobial activity at least against Rhizoctonia solani or Pyricularia oryzae, and shows the presence of components of about 70 kDa and/or about 65 kDa in molecular weight in the SDS-PAGE method. A gene encoding this protein and a method of using the same are provided.

Description

いもち病菌に対する完全生長阻害濃度は、３０μｇ全抽出タンパク／ｍｌ以下と概算され、このことから、ホンシメジの抽出物の中には高い抗菌活性をもつ物質が含まれていることが明らかとなった。このタンパク抽出液の菌に対する生長阻害の様式は、濃度の高い場合は発芽の完全抑制、低い場合は菌糸の生長阻害が観察された。菌糸の伸長阻害程度は、明らかに濃度依存的であった。また、いもち病菌の細胞は、その細胞質が細胞壁から離れ、原形質分離様の様相を呈した。
２）イオン交換カラムによる精製
次に抗菌タンパク質の精製を行った。ホンシメジ７０ｇを液体窒素中で粉砕し、２００ｍｌの緩衝液（５０ｍＭ ＭＥＳ，５０ｍＭ ＮａＣｌ，ｐＨ６．０）中で３０分間タンパクを抽出した。二重のミラクロスで濾過した後、１５０００×ｇで２０分間遠心し、不純物を沈殿させた。上清を濾紙でさらに濾過し、タンパク試料とした。タンパク試料約２００ｍｌを、イオン交換体Ｑ−ＳｅｐｈａｒｏｓｅＦＦ（Ｐｈａｒｍａｃｉａ社製）を充填したカラム（内径１．１ｃｍ×２０ｃｍ）にかけた。流速は２．５ｍｌ／分とし、基本緩衝液は５０ｍＭ Ｍｅｓ ｐＨ６．０， ５０ｍＭ ＮａＣｌ、溶出緩衝液は５０ｍＭ Ｍｅｓ ｐＨ６．０， １Ｍ ＮａＣｌを用い、グラジェント（ＮａＣｌで５０ｍＭから１Ｍ）は、試料を打った１００分後から１２０分間かけた。この後さらに溶出緩衝液を４０分間流した。画分の回収は、グラジェント開始から４０分ごとに４回行った（１００ ｍｌ／画分）。これら４種の画分（Ｉ，ＩＩ，ＩＩＩ，ＩＶ）の希釈系列を作製して、イネいもち病菌に対する抗菌アッセイを行った。その結果、画分ＩＩ〜ＩＶに抗菌活性が認められた。これらの画分によって病原菌細胞には原形質分離が生じた。最も活性の高かった画分ＩＩ（０〜３３３ｍＭ ＮａＣｌに相当）をセントリプレップ１０（Ａｍｉｃｏｎ社製、ＭＷＣＯ１０，０００）で濃縮し、イオン交換カラムＭｏｎｏＱＨＲ５／５（Ｐｈａｒｍａｃｉａ社製）にかけ、抗菌タンパクを部分精製した。流速は１ｍｌ／分とし、基本緩衝液は５０ｍＭ Ｍｅｓ ｐＨ６．０， ５０ｍＭ ＮａＣｌ、溶出緩衝液は５０ｍＭ Ｍｅｓ ｐＨ６．０， １Ｍ ＮａＣｌを用い、グラジェント（ＮａＣｌで５０ｍＭから１Ｍ）は、試料を打った２０分後から４０分間かけた。各画分（１ｍｌ）の一部をいもち病菌に対する抗菌アッセイと、ＳＤＳ−ＰＡＧＥ電気泳動に供試した。まず、ＨＰＬＣのチャートと抗菌性の強さとの関係を図１に示した。抗菌性はＭｏｎｏＱの各画分から５、１、０．２μｌをとり、いもち病菌に対するアッセイを行い、＋＋＋（０．２μｌまで阻害）、＋＋（１μｌまで阻害）、＋（５μｌまで阻害）、−（５μｌでも阻害しない）の４段階で表示した。その結果、Ａ２８０と、いもち菌に対する抗菌活性によりタンパクをモニターすると、ｐＨ６．０においてイオン強度（ＮａＣｌ濃度）が２５０ｍＭ付近に抗菌タンパクの溶出ピークが現れた。その後、イオン強度が高くなるにつれ、単位溶出液あたりのタンパク質の抗菌性は徐々に減少することが明らかとなった。
次に各画分から１０μｌをとり、等量の２×ＳＤＳ泳動用緩衝液（Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．１９８９）を加え、９５℃で５分間処理した後、Ｌａｅｍｍｌｉ（１９７０）の方法に従い、ＳＤＳ−ＰＡＧＥ電気泳動を行った。ゲルは１５％のＰＡＧＥＬ（ＡＴＴＯ社製）を用い、銀染色ＩＩキットワコー（和光純薬社製）によりタンパク質を検出した。タンパクのおよその分子量と量を見積もるため、分子量マーカー（ＬＭＷ：Ｐｈａｒｍａｃｉａ ＬＫＢ社製、分子量の大きい順に９４ｋＤａ、６７ｋＤａ、４３ｋＤａ、３０ｋＤａ、２０．１ｋＤａ、１４．４ｋＤａ）を１本のバンドが２０ｎｇとなるように泳動した。電気泳動像と抗菌性の強さの関係を図２に示した。各レーンの上に表示した数字は、図１の画分の番号と同じもので、抗菌性の強さも図１に準じて表示した。抗菌性とリンクすると考えられるタンパクのバンドを精査すると、約７０ｋＤａと、約６５ｋＤａの２つのバンドが候補として挙げられた（図２矢印）。これら２種のバンドの濃度と抗菌活性の度合いは正の相関関係にあるので、これらのバンドが抗菌タンパク質本体である可能性が強く示唆された。このうち、特に６５ｋＤａタンパクは、Ｑ−Ｓｅｐｈａｒｏｓｅによる分画においても、ＭｏｎｏＱによる分画においても、タンパクのバンドの濃度と、抗菌活性の強さとが非常によくリンクしていた。分子量マーカー（６７ｋＤａのアルブミン）から、デンシトメーターにより抗菌タンパクの量を推定し、いもち病菌に対する完全生長阻害濃度を算出すると、およそ５ｎｇ／ｍｌとなった。
３）抗菌性タンパク質のＮ末アミノ酸配列の解読
上記のｍｏｎｏＱの画分番号３６〜４４をセントリカットＶ−２０（クラボウ社製）で濃縮し、ＳＤＳ−ＰＡＧＥ電気泳動した。トリスを除去し、グリシンを含まない緩衝液系でＰＶＤＦ膜（ミリポア社製）へ転写し、クマシーブリリアントブルーＲ−２５０で軽く染色した後、脱色した。その後、抗菌性タンパク質であると考えられた７０ｋＤａと、６５ｋＤａのタンパクバンドを切り出した。Ｎ末端アミノ酸配列は、気相プロテインシークエンサー（ＨＰＧ１００５Ａ Ｐｒｏｔｅｉｎ Ｓｅｑｕｅｎｃｉｎｇ Ｓｙｓｔｅｍ）を用いて、エドマン分解法により決定した。
その結果、６５ｋＤａタンパクからは次のような３０アミノ酸
Ｎ末端−ＮＡＥＥＧＴＡＶＰＹＶＰＧＹＨＫＫＮＥＩＥＦＱＫＤＩＤＲＦＶ−Ｃ末端（配列番号３）
が決定された。
一方、７０ｋＤａタンパクは解読が不可能であった。この原因として、Ｎ末端がブロックされていることが考えられた。そこで、リシルエンドペプチダーゼ、Ｖ８プロテアーゼを用いて、７０ｋＤａタンパク質を部分消化し、４３ｋＤａリシルエンドペプチダーゼ消化物、４５ｋＤａＶ８プロテアーゼ消化物を得た。これらの部分消化タンパク質のアミノ酸配列を再解析した結果、前者から２４残基Ｎ末端−ＥＦＤＥＳＩＲＨＴＬＶＬＲＳＬＱＤＡＹＫＤＲＱＲ−Ｃ末端（配列番号４）
後者から２９残基
Ｎ末端−ＡＥＲＬＩＧＴＳＴＫＥＦＤＥＳＩＲＨＴＬＶＬＲＳＬＱＤＡＹ−Ｃ末端（配列番号５）
が決定された。両アミノ酸配列は大部分が重複していたので、部分分解による内部アミノ酸配列は、全体で３４残基
Ｎ末端−ＡＥＲＬＩＧＴＳＴＫＥＦＤＥＳＩＲＨＴＬＶＬＲＳＬＱＤＡＹＫＤＲＱＲ−Ｃ末端（配列番号６）
が決定されたことになる。決定されたアミノ酸配列に関して、データベースサーチを行ったところ、６５ｋＤａタンパクからの３０アミノ酸、７０ｋＤａタンパクからの３４アミノ酸は、ともにカワラタケのピラノースオキシダーゼと相同性を示した。そこで、ＭｏｎｏＱの各画分のピラノースオキシダーゼ活性を測定した。測定法は、Ｎｉｓｈｉｍｕｒａ ｅｔ ａｌ（１９９６）の方法に準じた。その結果、ピラノースオキシダーゼ活性の高さと、抗菌活性の強さはよく一致した。従って、抗菌性は、培地中のグルコースが本酵素によって酸化される際に生じる過酸化水素に由来すると推定された。次に、６５ｋＤａと７０ｋＤａの両タンパク質のみを含む画分（図２ 番号４２〜４４）を濃縮し、ピラノースオキシダーゼの諸性質を解析した。その結果、ホンシメジ由来抗菌タンパク質のピラノースオキシダーゼ活性は非常に高く、グルコースや、１，５−アンヒドログルシトールに対するＫｍ値も低いことが明らかとなった（表２）。

＊ １Ｕ＝１μｍｏｌｅ Ｈ_２Ｏ_２／分 ｐＨ７．０中、３７℃
酵素タンパク量（６５ｋＤａ＋７０ｋＤａ）は、ＳＤＳ−ＰＡＧＥ銀染色により定量した。
実施例３ ｃＤＮＡの単離
１）縮重プライマーの設計
１）で決定されたアミノ酸配列を基に、考えられる全ての塩基がミックスされたプライマーを合成した（Ｔｍは５２〜５６℃、括弧内の数字は縮重度を示す）。具体的には、６５ｋＤａタンパク由来のアミノ酸配列（３０残基）から以下の３種のプライマーを合成した。
６５Ｒ１（５’−ｇａｒｇａｒｇｇｉａｃｉｇｃｉｇｔｉｃｃ−３’（４））（配列番号７）
６５Ｒ２（５’−ｇａｒｔｔｙｃａｒａａｒｇａｙａｔｈｇａｙｍｇ−３’（３８４））（配列番号８）
６５Ｒ３（５’−ｔｔｙｇｔｉａａｙｇｔｉａｔｈｔｇｙｇｇｉｇｃ−３’（２４））（配列番号９）
一方、７０ｋＤａタンパクの部分分解物由来のアミノ酸配列（３４残基）からも、以下の３種のプライマーを合成した。
７０Ｆ１（５’−ｔｇｉｃｋｄａｔｉｓｗｙｔｃｒｔｃｒａａｙｔｃ−３’（３８４））（配列番号１０）
７０Ｆ２（５’−ｔｇｉｃｋｒｔｃｙｔｔｒｔａｉｇｃｒｔｃｙｔｇ−３’（６４））（配列番号１１）
７０Ｆ３（５’−ｇｇｉｇｃｒａａｄａｔｉｃｋｙｔｇｉｃｋｒｔｃ−３’（９６））（配列番号１２）
上記プライマー中、ｒはａまたはｇを、ｙはｃまたはｔを、ｈはａまたはｃまたはｔを、ｍはａまたはｃを、ｋはｇまたはｔを、ｄはａまたはｇまたはｔを、ｓはｇまたはｃを、ｗはａまたはｔを、ｉはイノシンをそれぞれ示す。
２）ホンシメジ子実体ｃＤＮＡライブラリーの構築
ホンシメジ子実体からＳＤＳフェノール法で全核酸を抽出し、塩化リチウム沈殿により全ＲＮＡを回収した。これからｍＲＮＡ ｐｕｒｉｆｉｃａｔｉｏｎ ｋｉｔ（Ｐｈａｒｍａｃｉａ社製）によりホンシメジｍＲＮＡを調製した。子実体約１０ｇから２０μｇのｍＲＮＡが得られた。このうち５μｇをＺＡＰ ｃＤＮＡ ｓｙｎｔｈｅｓｉｓ ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ社製）に供試し、ｃＤＮＡを合成した。ゲル濾過カラムにより１〜５ｋｂのｃＤＮＡを分画し、Ｕｎｉ−ＺＡＰ ＸＲベクター（Ｓｔｒａｔａｇｅｎｅ社製）にライゲーションし、Ｇｉｇａｐａｃｋ ＩＩＩ（Ｓｔｒａｔａｇｅｎｅ社製）によりパッケージングを行った。全ての手順はキット添付の説明書に従った。構築したホンシメジｃＤＮＡライブラリーのタイターは、およそ３００万ｐｆｕと算出された。
３）ＲＴ−ＰＣＲによるプローブの取得
１）で合成したプライマーを用いて２）で合成したｃＤＮＡを鋳型にＰＣＲを行い、ライブラリーをスクリーニングするためのプローブとなるホンシメジタンパクの部分長ｃＤＮＡの増幅を試みた。反応条件は以下のように行った。５０μｌの反応液中に、ｃＤＮＡ １００ｎｇ，１０_×Ｅｘ ｔａｑ ｂｕｆｆｅｒ ５μｌ，ｄＮＴＰｓ ４μｌ，ｐｒｉｍｅｒ １０ｐｍｏｌｅｓ／ｅａｃｈ ｋｉｎｄ ｏｆ ｓｅｑｕｅｎｃｅ，Ｅｘ ｔａｑ（Ｔａｋａｒａ社製）＋Ｔａｑ ＳＴＡＲＴ ａｎｔｉｂｏｄｙ（Ｃｌｏｎｔｅｃｈ社製）１μｌをそれぞれ含み、プログラムテンプコントロールシステムＰＣ−７００（ＡＳＴＥＫ社）を用いて、９４℃ ３分を１回、９４℃ １分、５０℃ １分、７２℃ １分を３５回、７２℃ ６分を１回行った。その結果、６５Ｒ１−７０Ｆ１、６５Ｒ１−７０Ｆ２、６５Ｒ２−７０Ｆ１、６５Ｒ２−７０Ｆ２、６５Ｒ２−７０Ｆ３、６５Ｒ３−７０Ｆ１のプライマー組み合わせにおいて約０．４〜０．５ｋｂの産物が増幅された。これらのうち増幅効率のより高かった６５Ｒ２−７０Ｆ１の組み合わせによる約０．４ｋｂの断片をゲル精製し、ベクターｐＣＲＩＩ（Ｉｎｖｉｔｒｏｇｅｎ社製）にクローニングし、塩基配列を決定した。この塩基配列から推定されるアミノ酸配列は、２３）で決定されたアミノ酸配列の一部と同一の配列を含み、しかも配列全体に渡ってカワラタケのピラノースオキシダーゼと緩い相同性を示した。このことから、精製した７０ｋＤａと６５ｋＤａの抗菌タンパク質は、一つの遺伝子にコードされ、ＲＴ−ＰＣＲにより得られたｃＤＮＡクローンはホンシメジ抗菌タンパク質の部分長ｃＤＮＡであることが確認された。
４）完全長ｃＤＮＡのスクリーニング
３）で得られたクローンをベクターから切り出し、これをプローブに用いて２）で構築したホンシメジｃＤＮＡライブラリーをスクリーニングした。１４×１０ ｃｍの角形シャーレにＺＡＰ ｃＤＮＡ ｓｙｎｔｈｅｓｉｓ ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ社製）の説明書に従って、約１５０００ｐｆｕのファージを宿主菌ＸＬ１−ｂｌｕｅ ＭＲＦ’とともにプレーティングした。プラークをＨｙｂｏｎｄ−Ｎ＋ナイロンメンブレンフィルター（Ａｍｅｒｓｈａｍ社製）に接触させ、メンブレン添付の説明書きに従ってアルカリ処理を行い、ＤＮＡを変性させ、メンブレン上に固定させた。ハイブリダイゼーション、および洗浄の条件は、メンブレンに添付の説明書きに従って高いストリンジェンシー下で行った。１次スクリーニングで約１２０，０００ｐｆｕのファージから２０個のポジティブクローンが得られた。これらのクローンに関して、２次スクリーニング、プラークの精製を兼ねた３次スクリーニングを行い、２０個全てについてＺＡＰ ｃＤＮＡ ｓｙｎｔｈｅｓｉｓ ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ社製）の説明書きに従って、ｉｎ ｖｉｖｏ ｅｘｃｉｓｉｏｎを行った。その結果、１８個のクローンが、ファージミドベクターｐＢｌｕｅｓｃｒｉｐｔ ＳＫに組み込まれたｃＤＮＡとして回収された。これらのクローンの長さは１．７〜２．１ｋｂであり、制限酵素分析から非常によく似た遺伝子由来であることが示唆された。
５）塩基配列の決定
上記の１８個のｃＤＮＡクローンについて、５’および３’側の塩基配列およそ５００ｂｐを決定した。得られた塩基配列データをＧｅｎｅｔｙｘ ｖｅｒ．９．０解析ソフト（Ｓｏｆｔｗａｒｅ Ｄｅｖｅｌｏｐｍｅｎｔ社製）を用いて解析した。その結果、ポリＡ付加部位にはクローン間で差異があるものの、全てのクローンが２３）で決定した６５ｋＤａタンパクの３０アミノ酸をコードするＤＮＡ配列を含んでいた。番号１３（２．１ｋｂ）のｃＤＮＡクローンが最長であったので、このクローンの全塩基配列を決定した。プライマーウォーキング法により、ＡＢＩ ＰＲＩＳＭ蛍光シークエンサー（Ｍｏｄｅ１３１０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ，Ｐｅｒｋｉｎ Ｅｌｍｅｒ社製）を用いて塩基配列を決定した。その結果、ホンシメジ抗菌タンパクをコードするｃＤＮＡは全長２１０６塩基対からなり、１８５４塩基対のオープンリーディングフレームを含み、６１８個のアミノ酸をコードしていた（配列番号１および２）。アミノ酸配列から推定される分子量は約６８４８７、等電点は６．１２と算出された。精製タンパクから決定されたアミノ酸配列は、６５ｋＤａ由来３０アミノ酸が、配列表配列番号２のアミノ酸番号７６〜１０５に、７０ｋＤａ由来３４アミノ酸が、同２１１〜２４４にそれぞれ対応した。これは６５ｋＤａと７０ｋＤａタンパクが同じ一つの遺伝子にコードされていることを意味する。さらに推定糖鎖付加部位が７カ所（配列表配列番号２のアミノ酸番号１５４、３１９、３６０、４１２、５５８、５７３、５８３）存在していた。
以上の結果から、クローニングしたｃＤＮＡはホンシメジ抗菌タンパクをコードする遺伝子に由来すると結論された。本発明のホンシメジ由来抗菌タンパク質のアミノ酸配列について、データベース（ＤＤＢＪ）上で相同性検索（ＢＬＡＳＴ）を実施したところ、カワラタケのピラノースオキシダーゼのアミノ酸配列と全体で４５％の同一性を有していた。これ以外で有意に相同性のある配列は存在していないことから、本遺伝子は新規なピラノースオキシダーゼ様タンパク質をコードする遺伝子であると考えられる。
実施例４．大腸菌内発現と組換えタンパク質の精製
１）発現ベクターの構築
実施例３の５）で単離した１３番のｃＤＮＡクローンには、翻訳終止コドンの約０．０６ｋｂ下流に唯一のＥｃｏＴ２２Ｉ制限部位が、また翻訳終止コドンの約０．２５ｋｂ上流に唯一のＢａｍＨＩ制限部位が存在する。またこのｃＤＮＡの５’側のベクター上のマルチクローニングサイトには、ＢａｍＨＩが存在する。そこで、まずこのｃＤＮＡが組みこまれたプラスミド（ベクターはｐＢｌｕｅｓｃｒｉｐｔ）を制限酵素ＥｃｏＴ２２Ｉ（Ｔａｋａｒａ社製）で完全消化し、その後ＢａｍＨＩ（Ｔａｋａｒａ社製）で部分消化した。その結果生じた約２ｋｂのＢａｍＨＩ（ベクター上のＢａｍＨＩ）−ＥｃｏＴ２２Ｉ断片を、予め制限酵素ＢａｍＨＩ、ＰｓｔＩで二重消化した大腸菌用発現ベクターｐＱＥ３０（Ｑｉａｇｅｎ社製）へ組みこんだ（ｐＱＥＨＳＰＯｆｕｌｌと命名）。完成した構築物は、本発明のホンシメジピラノースオキシダーゼ様タンパクをコードする完全長ｃＤＮＡの最長オープンリーディングフレーム（ＯＲＦ）を含み（配列表の配列番号１の第８番目から第１８６４番目の塩基配列を含む。これは配列表の配列番号２の全アミノ酸配列をコードする）、発現タンパク質のＮ末端にはタグとして６個のヒスチジン残基が付加される。
２）大腸菌内発現
宿主大腸菌Ｍ１５株を用いて発現実験を行った。菌の培養方法、ＩＰＴＧ（Ｉｓｏｐｒｏｐｙｌ β−Ｄ−Ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ）によるタンパク発現誘導方法はＱｉａｇｅｎ社の説明書に従った。抗生物質アンピシリン、カナマイシンを含むＬＢ培地中でＯＤ_６００＝０．５程度になるまで菌を前培養し、その後同じ培地中で各種温度、各種ＩＰＴＧ濃度にて一定時間培養し発現誘導を行った。菌体からＱｉａｇｅｎ社の説明書に従って可溶性タンパク質を抽出し、そのうちの一定量をＮｉｓｈｉｍｕｒａ ｅｔ ａｌ． （１９９６）の方法に従ってピラノースオキシダーゼ活性を測定した。組換えタンパク質の発現結果を表３にまとめた。

まず最初に通常の誘導条件である培養温度３７℃、ＩＰＴＧ濃度２ｍＭで誘導を試みたが、不溶性封入体は大量に発現したものの、可溶性画分のピラノースオキシダーゼ活性が検出されなかった。そこで種々の条件で発現誘導を行い、可溶性画分のピラノースオキシダーゼ活性を測定した。その結果、誘導条件を緩くするににつれ、即ち培養温度やＩＰＴＧ濃度を下げていくにつれピラノースオキシダーゼ活性が上昇した。これは誘導条件の緩和により、可溶性組換えタンパク質含量が増加したためと考えられた。一方、対照としたｐＱＥ３０（ベクターのみ）では活性は全く検出されなかった。以上の結果からクローニングしたｃＤＮＡは確かに活性のあるピラノースオキシダーゼ様タンパク質をコードしていることが明らかとなった。なお、培養温度２５℃、ＩＰＴＧ濃度０．５ｍＭでは５時間の培養に比べ、２１時間での培養は活性が下がった。これはおそらく発現タンパク質が分解されていることを示唆している。
次に発現タンパク質の精製を試みた。培養温度１６℃、ＩＰＴＧ濃度０．１ｍＭで発現誘導した菌体由来の可溶性タンパク質画分から、Ｎｉ−ＮＴＡアガロース（Ｑｉａｇｅｎ社製）を用いて組換えピラノースオキシダーゼ様タンパク質の精製を行った。Ｑｉａｇｅｎ社の説明書に従って、タンパク質の吸着、洗浄、溶出を行ったところ、溶出画分にのみピラノースオキシダーゼ活性が検出された。従って、組換えタンパク質のＮ末端は大腸菌内で分解されておらず、ヒスチジン残基がＮ末に結合していること、それを利用して組換えタンパク質をＮｉ−ＮＴＡアガロースによって容易に精製出来ること、またクローニングした完全長ｃＤＮＡの全コード領域は、そのままで（タンパクのＮ末端側に相当する部分を除去しなくても）活性のあるタンパク質をコードしていることが明らかとなった。組換えタンパク質の収量は大腸菌培養液１１当たり数ｍｇと推定された。
効果
本発明の配列表の配列番号２の配列または、このうち第１番目から第７５番目の配列を除いた全配列を含むことを特徴とするタンパク質成分を有効成分として含む製剤を作出すれば、強力な抗菌剤としての利用が期待できる。また、上記タンパク質成分を有効成分として含む試薬を作出すれば、血糖など、糖の測定に利用することが出来る。本発明の配列表の配列番号１のＤＮＡ配列のうち、第８番目から第１８６４番目の配列であることを特徴とするＤＮＡ配列、または第２３３番目から第１８６４番目の配列であることを特徴とするＤＮＡ配列を、植物細胞の中で機能しうる適当な構成的、器官・時期特異的、あるいはストレスや病害虫で誘導されるプロモーター配列と、植物細胞で機能しうるターミネター配列の発現カセットに組み込み、植物細胞に導入、再生個体を得ることにより、病害虫抵抗性植物を作出できることが期待される。あるいはまた、上記のＤＮＡ配列を大腸菌や酵母あるいは昆虫やある種の動物細胞に、それぞれの宿主で増幅可能な発現ベクターを用いて導入、発現させることにより、当該タンパクを大量に安価に得ることができる。
【配列表】

【図面の簡単な説明】
図１は、ＭｏｎｏＱカラムによるホンシメジタンパク質の分離のチャートと抗菌性との関係を示す。
図２は、ＭｏｎｏＱカラムによるホンシメジ抗菌タンパク質の分離の電気泳動像と抗菌性との関係を示す。レーン上の番号は図１の画分番号に一致し、Ｍは分子量マーカーを示す。またレーン下の記号（−、＋、＋＋、＋＋＋）は抗菌活性の強さを示す。抗菌タンパク（７０ｋＤａと６５ｋＤａ）を矢印で示した。 Technical field
The present invention relates to a novel protein having antibacterial activity, a gene encoding the protein, and a method for using the protein and the gene. More specifically, the present invention relates to a hon-shimeji-derived protein having antibacterial activity against at least rice blast fungus and rice blast fungus, a gene encoding the protein, and a method of using the protein and the gene.
This application is a priority claim application based on Japanese Patent Application No. 11-267238 filed on Sep. 21, 1999. The contents of the Japanese patent application are all incorporated herein.
Background art
As plant proteins having antifungal activity or antibacterial activity against plant pathogens, lytic enzymes such as chitinase and β-1,3-glucanase are conventionally known. In in vitro experiments, these enzymes are effective alone (Schlumbaum et al. (1986), Nature 324, p. 365-367; Brogley et al. (1991), Science 254, p. 1194-1197), but generally 2 It is known that higher effects can be obtained by using a combination of more than one enzyme (Mouch et al. (1988), Plant Physiol. 88, p. 936-942; Sela-Burage et al. (1993), Plant Physiol. 101, 857-863). The concentration of these lytic enzymes to suppress the growth of filamentous fungi generally requires about several tens to several hundreds μg / ml when used alone, or about several μg / ml per enzyme even when combined. It has been known. However, among these lytic enzymes, rice blast fungus, which is an important disease of rice (Pyricularia oryzaeNothing that has been proved to be antibacterial has been reported to the extent that the inventor knows.
On the other hand, small molecular weight AFP (anti-fungal peptide) including defensin also has antimicrobial activity. Of these, rice blast fungus (Pyricularia oryzae) And blight fungus (Rhiz octonia solani) As an antibacterial agent for both of Ca-AMP1 (Japanese Patent Publication No. 8-505048) and CB-1 (Oita et al. (1996), Biosci. Biotech. Biochem. 60, p. 481-483). However, Rs-AFP1 and Rs-AFP2 (Terras et al. (1992), J. Biol. Chem. 267, p. 15301-15309), and Ace- are considered to have antibacterial activity against Pyricularia oryzae. AMP1 (Tokuheihei 9-501424) has been reported. These low molecular weight peptides inhibit the growth of various phytopathogenic bacteria including the above pathogenic bacteria by 50% at a few μg / ml.
Attempts have also been made to isolate genes for lytic enzymes or low molecular weight antibacterial peptides and introduce them into plants to produce disease resistant plants (Broglie et al. (1991), Science 254, p. 1194-1197; Zhu et al.). (1994), Bio / Technology 12, p.807-812; Lin et al. (1995), Bio / Technology 13, p.686-691; Terras et al. (1995), The Plant Cell 7, p.573-588). . However, under the present circumstances, a plant body imparted with a practical level of resistance is not always obtained. The reason for this is that the expression level of the introduced gene is low, but more essentially, it is considered that the antibacterial activity of the antibacterial protein itself reported so far is low. Therefore, it is desired to identify and utilize a stronger antibacterial protein than conventional antibacterial proteins.
Summary of the Invention
One of the objects of the present invention is to search for and identify a novel antibacterial protein capable of suppressing the growth of various plant pathogens including rice blast fungus and blight fungus which are two major diseases of rice at relatively low concentrations. That is.
Another object of the present invention is to clone a gene encoding the above novel protein and specify the base sequence thereof.
Still another object of the present invention is to introduce a gene of the present invention into a host organism (microorganism, animal, plant, etc.) to produce a transformant and to utilize the gene of the present invention.
Yet another object of the present invention is to provide an antimicrobial agent comprising the antimicrobial protein of the present invention.
Detailed Description of the Invention
In order to solve the above-mentioned problems, the present inventors first established an assay system for assaying in vitro antibacterial activity against blast fungus and blight fungus. Next, the present inventors extract proteins from hon-shimeji mushroom, a kind of edible mushrooms, combine ion exchange column chromatography and high performance liquid chromatography (HPLC), and test each fraction in the above assay system. The antibacterial protein fraction was identified, and the antibacterial protein was isolated and purified. Furthermore, the present inventors determined a partial amino acid sequence of the purified protein, and obtained a partial length cDNA encoding the protein by RT-PCR using an oligonucleotide synthesized based on this amino acid sequence as a primer. Next, the present inventors isolated a full-length cDNA encoding the protein by screening a cDNA library derived from hon shimeji using this partial length cDNA as a probe, and determined the entire nucleotide sequence. As described above, the present inventors succeeded in the isolation of a novel antibacterial protein derived from hon-shimeji mushroom and the cloning of the DNA encoding the same, and the amino acid sequence of the protein and the base sequence of the DNA were determined. It came to complete.
That is, according to the first aspect of the present invention, it can be obtained from a fraction precipitated by an ammonium sulfate precipitation method from an aqueous extract of hon-shimeji mushroom, has at least antibacterial activity against rice blight fungus or rice blast fungus, and SDS. An antimicrobial protein is provided that has a molecular weight of about 70 kDa in the precursor form and about 65 kDa in the mature form by the PAGE method.
The antimicrobial protein of the present invention typically has the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing. Without limitation, the mature form of about 65 kDa is assumed to consist of amino acid residues 76-618 of SEQ ID NO: 1.
The antibacterial protein of the present invention has not only the amino acid sequence shown in SEQ ID NO: 2, but also an amino acid sequence having one to a plurality of amino acid mutations in the sequence or an amino acid sequence having 50% or more homology with this sequence. In addition, an antibacterial protein exhibiting antibacterial activity against rice blast fungus or rice blast fungus is also included.
The antibacterial protein of the present invention is preferably 60% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, most preferably the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing. It has an amino acid sequence having a homology of 95% or more.
According to the second aspect of the present invention, a polypeptide comprising a partial amino acid sequence of SEQ ID NO: 2 in the sequence listing, for example, amino acid residues 76-618; A polypeptide having an amino acid mutation and a polypeptide having an amino acid sequence having 50% or more homology with any of the amino acid sequences, wherein the polypeptide exhibits antibacterial activity against rice blast fungus or rice blast fungus; Antibacterial proteins are provided that consist of a single or any combination of polypeptides.
Regarding the antibacterial protein according to the second aspect of the present invention, the definition of “a protein having 50% or more homology” with each specific amino acid sequence should have at least 50% or more homology. Is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more. Is done.
According to a third aspect of the present invention, the step of recovering the fraction precipitated by the sulfuric acid precipitation method using ammonium sulfate 75% saturation from the aqueous extract of hon-shimeji;
Subjecting the fraction to ion exchange chromatography and collecting a fraction eluted at a NaCl concentration of 0.05 M to 1 M;
A method for producing the antibacterial protein of the present invention is provided.
According to the fourth aspect of the present invention, a gene encoding the antibacterial protein of the present invention is provided.
The gene of the present invention typically has a base sequence set forth in SEQ ID NO: 1 in the sequence listing, a base sequence having one to a plurality of base substitutions, deletions, insertions and / or additions in the base sequence, Alternatively, it has a base sequence that hybridizes with the above base sequence under stringent conditions.
The gene of the present invention is generally 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably the nucleotide sequence shown in SEQ ID NO: 1 of the Sequence Listing. It has a base sequence having a homology of 90% or more, most preferably 95% or more.
According to a fifth aspect of the present invention, there is provided an oligonucleotide for obtaining an antibacterial protein derived from hon-shimeji mushroom,
Two regions are selected from the base sequence of the gene encoding the antibacterial protein shown in SEQ ID NO: 1 in the sequence listing so as to satisfy the following conditions:
1) The length of each region is 15-30 bases;
2) The percentage of G + C in each region is 40-60%;
A single-stranded DNA having the same base sequence as the above region or a base sequence complementary to the above region is produced, or the genetic code is degenerate so as not to change the amino acid residue encoded by the single-stranded DNA. Producing a mixture of single-stranded DNAs considered, and if necessary, producing the single-stranded DNA modified so as not to lose the binding specificity to the base sequence of the gene encoding the antibacterial protein
The oligonucleotide manufactured by the method including this is provided.
The oligonucleotide of the present invention preferably has the nucleotide sequence described in any one of SEQ ID NOs: 7 to 12 in the sequence listing.
According to the sixth aspect of the present invention, an amplification reaction is carried out using a hon-shimeji cDNA library as a template using the two sets of oligonucleotides as primers, and a part of the gene encoding the antibacterial protein of the present invention is selected. There is provided a method for isolating a gene of the present invention, comprising amplifying and screening the cDNA library using the obtained amplification product as a probe to isolate a full-length cDNA clone.
According to the seventh aspect of the present invention, a recombinant vector comprising the gene of the present invention is provided.
In the recombinant vector of the present invention, preferably the vector is an expression vector.
According to the eighth aspect of the present invention, there is provided a transformant obtained by introducing the recombinant vector of the present invention into a host organism.
According to the ninth aspect of the present invention, there is provided an antibacterial agent comprising the antibacterial protein of the present invention as an active ingredient.
Hereinafter, preferred embodiments will be described in detail for the purpose of explaining the present invention.
According to the first aspect of the present invention, a hon-shimeji-derived protein having an antibacterial effect against phytopathogenic fungi is provided. As long as the protein of the present invention has the characteristics described in the present specification, its origin, production method and the like are not limited. That is, the antibacterial protein of the present invention may be any of naturally occurring proteins, proteins expressed from recombinant DNA by genetic engineering techniques, or chemically synthesized proteins.
The protein of the present invention typically has a 618 amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. However, among natural proteins, there are one to more than one due to differences in the varieties of species that produce them, gene mutations due to differences in ecotypes, or the presence of similar isozymes. It is well known that mutant proteins having amino acid mutations exist. As used herein, the term “amino acid mutation” means substitution, deletion, insertion and / or addition of one or more amino acids. The protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 2 based on the assumption from the base sequence of the cloned gene, but is not limited to the protein having the sequence. It is intended to include all homologous proteins as long as they have the properties described in. The homology is at least 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.
As used herein, the percent homology is determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). Is possible. The program can be used on the Internet from the website of National Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ). Various conditions (parameters) for homology search by the BLAST program are described in detail on the same site, and some settings can be appropriately changed, but the search is usually performed using default values.
In general, substitution between amino acids having similar properties (for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid, substitution from one acidic amino acid to another When a substitution to an acidic amino acid or a substitution from one basic amino acid to another basic amino acid is introduced, the resulting mutant protein often has the same properties as the original protein. Techniques for producing recombinant proteins having such desired mutations using gene recombination techniques are well known to those skilled in the art, and such mutant proteins are also included within the scope of the present invention.
The protein of the present invention has an SDS-PAGE molecular weight of about 70 kDa in a precursor form (corresponding to a polypeptide comprising the first to 618th amino acid sequences in the sequence of SEQ ID NO: 2 in the sequence listing), and a mature form ( (Corresponding to a polypeptide consisting of the 76th to 618th amino acid sequences of the sequence number 2 in the sequence listing) is about 65 kDa. Although not necessarily limited, the antibacterial protein of the present invention typically has the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
Therefore, according to the present invention, a polypeptide consisting of the first to 618th amino acid sequences of SEQ ID NO: 2 in the sequence listing; and a polypeptide consisting of the 76th to 618th amino acid sequences alone or any of them An antibacterial protein comprising the combination of: In addition, the said polypeptide includes the homologous polypeptide which has a variation | mutation as described above in this specification.
The protein of the present invention can be purified from hon-shimeji fruiting bodies using, for example, ammonium sulfate precipitation and ion exchange column chromatography according to the examples described later. Alternatively, among the DNA sequences of SEQ ID No. 1 in the sequence listing according to the present invention, a DNA sequence consisting of the 8th to 1861st sequence, or a DNA sequence consisting of the 233rd to 1861st sequence, Alternatively, a large amount of the protein can be obtained by introducing and expressing an insect or certain animal cells using an expression vector that can be amplified in each host.
When the homology search by BLAST of DDBJ is conducted for the antibacterial protein derived from hon-shimeji mushroom according to the present invention, it has the highest homology and has only 45% homology between the entire amino acid sequences. Since a certain sequence does not exist, this protein is considered to be a novel protein. If the amino acid sequence of this protein and the DNA sequence encoding the protein are disclosed by the present invention, using the sequence or a part thereof, a genetic engineering technique such as hybridization or nucleic acid amplification reaction such as PCR may be used. A gene encoding a protein having the same physiological activity can be easily isolated from other species. In such a case, novel proteins encoded by these genes are also included in the scope of the present invention. When the homology search of the DNA sequence of the present invention is performed using the BLAST program described above, only those having homology (93%) within a very short range (32 bases) are hit.
The antibacterial protein derived from the hon-shimeji mushroom of the present invention had the highest homology (45% of the entire amino acid sequence) was the pyranose oxidase of Corarius versicolor. Pyranose oxidase is an enzyme that oxidizes pyranose such as glucose and generates 2-keto product to generate hydrogen peroxide, and its application value to measurement of other pyranose is described. See JP-A-8-205861 incorporated herein. It was found that the hon-shimeji-derived antibacterial protein of the present invention actually showed pyranose oxidase activity, had a very high specific activity, and a low Km value for glucose and the like. Moreover, the strength of the antibacterial activity of the fraction obtained by the ion exchange column and the height of the pyranose oxidase activity were in good agreement. Therefore, the antibacterial activity of the hon-shimeji mushroom-derived antibacterial protein found in the present invention is presumed to be related to pyranose oxidase. Without being bound by theory, the mechanism of action of the antibacterial activity in the present invention may be that hydrogen peroxide generated in the process of converting glucose contained in the assay medium into the enzyme is harmful to pathogenic bacteria. It is done.
Purification and isolation of the protein of the present invention can be carried out by appropriately combining methods commonly used for protein purification and isolation such as ammonium sulfate precipitation and ion exchange chromatography (MonoQ, Q Sepharose, DEAE, etc.). it can.
For example, as described in the examples below, finely ground hon-shimeji mushrooms are extracted with a buffer and then filtered, and ammonium sulfate (ammonium sulfate) is added to the supernatant to a suitable concentration, for example, 75% saturation. The precipitate containing the protein of the present invention is obtained by allowing to stand. The precipitate may be further dialyzed, then subjected to ion exchange chromatography, and eluted with a salt concentration gradient (for example, 50 mM to 1 M with sodium chloride) to recover a fraction containing the desired protein.
The present invention also provides a gene encoding the antimicrobial protein of the present invention. The type of gene is not particularly limited, and may be any of naturally-derived DNA, recombinant DNA, and chemically synthesized DNA, and any of genomic DNA clones and cDNA clones.
The gene of the present invention typically has the base sequence set forth in SEQ ID NO: 1 in the sequence listing, but this is only a base sequence of the clone obtained in the following Examples, which is merely an example of the present invention. . Among natural genes, there are a small number of mutations due to differences in the varieties of species that produce them, differences in ecotypes, and the presence of similar isozymes. Well known to those skilled in the art. Therefore, the gene of the present invention is not limited to the gene having the base sequence described in SEQ ID NO: 1 in the Sequence Listing, but includes all genes encoding the antibacterial protein of the present invention.
In particular, if the amino acid sequence of this protein and the DNA sequence encoding it are disclosed by the present invention, using this sequence or a part thereof, a basic technique of genetic engineering such as hybridization or nucleic acid amplification reaction may be used. Thus, a gene encoding a protein having the same physiological activity can be easily isolated from other biological species. In such a case, such a gene is also included in the scope of the present invention.
Hybridization conditions used for screening for homologous genes are not particularly limited, but generally stringent conditions are preferable, for example, 6 × SSC, 5 × Denhardt's, 0.1% SDS, 25 ° C. to 25 ° C. It is conceivable to use hybridization conditions such as 68 ° C. In this case, the hybridization temperature is more preferably 45 ° C. to 68 ° C. (no formamide) or 25 ° C. to 50 ° C. (50% formamide). It is well known to those skilled in the art that DNA containing a base sequence having a homology more than a certain homology can be cloned by appropriately setting hybridization conditions such as formamide concentration, salt concentration and temperature. All homologous genes that have been made are included within the scope of the present invention.
Nucleic acid amplification reactions include, for example, replication chain reaction (PCR) (Saiki et al., 1985, Science 230, p. 1350-1354), ligase chain reaction (LCR) (Woo et al., 1989, Genomics 4, p. 560-569; Ballinger et al., 1990, Gene 89, p. 117-122; Barany et al., 1999, Proc. Natl. Acad. Sci. USA 88, p. 189-193) and transcription-based amplification (Cou et al., 1989, Proc. Natl. Acad.Sci.USA 86, p.1173-1177), as well as strand displacement reactions (SDA) (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89, p. 392-396; Walker et al., 1992, Nuc. ds.Res.20, p.1691-1696), self-retained sequence replication (3SR) (Guateri et al., 1990, Proc. Natl. Acad. Sci. USA 87, p, 1874-1878) and the Qβ replicase system (Rezildi et al. 1988, BioTechnology 6, pp. 1197-1202). In addition, amplification based on a nucleic acid sequence by competitive amplification of a target nucleic acid and a mutant sequence described in European Patent No. 0525882 (Nucleic Acid Sequence Based Amplification: NASABA) reaction or the like can also be used. The PCR method is preferred.
The homologous gene cloned using the above hybridization, nucleic acid amplification reaction or the like is at least 50% or more, preferably 60% or more, more preferably relative to the base sequence described in SEQ ID NO: 1 in the Sequence Listing. It has a homology of 70% or more, more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.
According to the present invention, there is also provided an oligonucleotide for obtaining an antibacterial protein derived from hon-shimeji mushroom,
Two regions are selected from the base sequence of the gene encoding the antibacterial protein shown in SEQ ID NO: 1 in the sequence listing so as to satisfy the following conditions:
1) The length of each region is 15-30 bases;
2) The percentage of G + C in each region is 40-60%;
A single-stranded DNA having the same base sequence as the above region or a base sequence complementary to the above region is produced, or the genetic code is degenerate so as not to change the amino acid residue encoded by the single-stranded DNA. Producing a mixture of single-stranded DNAs considered, and if necessary, producing the single-stranded DNA modified so as not to lose the binding specificity to the base sequence of the gene encoding the antibacterial protein
The oligonucleotide manufactured by the method including this is provided. The oligonucleotide of the present invention can be used for, for example, hybridization for detecting or isolating the gene of the present invention, or amplification reaction such as PCR using appropriate two types as primer pairs.
The oligonucleotide of the present invention preferably has a nucleotide sequence described in any one of SEQ ID NOs: 8 to 12 in the Sequence Listing. This nucleotide sequence is designed as a primer for PCR for cloning of a gene fragment encoding each protein based on the amino acid sequence of SEQ ID NO: 1. All nucleotides capable of encoding the amino acid Primer mixed with base.
A partial fragment of the gene of the present invention can be isolated by amplification by performing a nucleic acid amplification reaction such as PCR using a Hongjimji fruiting body cDNA library as a template using an appropriate combination of the above oligonucleotides. A full-length cDNA clone can be isolated by screening the cDNA library by plaque hybridization or the like using the amplification product thus obtained as a probe. Nucleic acid amplification reaction procedures and conditions, plaque hybridization conditions, and the like are well known to those skilled in the art.
The present invention also provides a recombinant vector containing the gene of the present invention. As a method for incorporating the DNA fragment of the gene of the present invention into a vector such as a plasmid, see, for example, Sambrook, J. et al. Et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1.53 (1989). For convenience, a commercially available ligation kit (for example, manufactured by Takara Shuzo Co., Ltd.) can also be used. The recombinant vector thus obtained (for example, a recombinant plasmid) is introduced into a host cell (for example, E-Coil TB1, LE392 or XL-1Blue).
As a method for introducing a plasmid into a host cell, Sambrook, J. et al. Et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1.74 (1989), chemistry such as calcium phosphate method / calcium chloride / rubidium chloride method, electroporation method, electroinjection method, PEG, etc. And a method using a general treatment, a method using a gene gun, and the like.
A vector can be conveniently prepared by ligating a desired gene to a recombination vector (for example, plasmid DNA) available in the art by a conventional method. Specific examples of the vector used include, but are not limited to, plasmids derived from Escherichia coli such as pBluescript, pUC18, pUC19, and pBR322.
In particular, expression vectors are useful for the purpose of producing a desired protein. The type of expression vector is not particularly limited as long as it has a function of expressing a desired gene and producing a desired protein in various prokaryotic and / or eukaryotic host cells. As expression vectors, pQE-30, pQE-60, pMAL-C2, pMAL-p2, pSE420 and the like are preferable. As expression vectors for yeast, pYES2 (genus Saccharomyces), pPIC3.5K, pPIC9K, pAO815 (genus Pichia), insects Preferred expression vectors for use include pBacPAK8 / 9, pBK283, pVL1392, and pBlueBac4.5.
A transformant can be prepared by introducing a desired expression vector into a host cell. The host cell to be used is not particularly limited as long as it is compatible with the expression vector of the present invention and can be transformed, and is a natural cell usually used in the technical field of the present invention or artificially established. Various cells such as recombinant cells can be used. Examples include bacteria (Escherichia, Bacillus), yeasts (Saccharomyces, Pichia, etc.), animal cells, insect cells, plant cells, and the like.
The host cell is preferably Escherichia coli, yeast or insect cell. Specifically, Escherichia coli (M15, JM109, BL21 etc.), yeast (INVSc1 (genus Saccharomyces), GS115, KM71 (above Pichia) etc.), insect cell ( BmN4, silkworm larvae, etc.) are exemplified. Examples of animal cells include mouse-derived, Xenopus-derived, rat-derived, hamster-derived, monkey-derived or human-derived cells, or cultured cell lines established from these cells. Furthermore, plant cells are not particularly limited as long as cell culture is possible, and examples thereof include cells derived from tobacco, Arabidopsis, rice, corn, wheat, and the like.
When a bacterium, particularly E. coli, is used as a host cell, the expression vector generally comprises at least a promoter / operator region, a start codon, a gene encoding a desired antibacterial protein, a stop codon, a terminator, and a replicable unit.
When yeast, plant cells, animal cells or insect cells are used as host cells, the expression vector generally contains at least a promoter, an initiation codon, a gene encoding a desired antibacterial protein, a stop codon, and a terminator. preferable. Further, it may appropriately contain a DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5 'side and 3' side of the desired gene, a selectable marker region or a replicable unit.
In the vector of the present invention, a suitable start codon is exemplified by a methionine codon (ATG). Moreover, as a stop codon, a common stop codon (for example, TAG, TGA, TAA etc.) is illustrated.
A replicable unit means a DNA having the ability to replicate its entire DNA sequence in a host cell, a natural plasmid, an artificially modified plasmid (a plasmid prepared from a natural plasmid) and Synthetic plasmids and the like are included. Suitable plasmids include E. coli. In plasmid, plasmid pQE30, pET or pCAL or an artificially modified product thereof (DNA fragment obtained by treating pQE30, pET or pCAL with an appropriate restriction enzyme), plasmid pYES2 or pPIC9K in yeast, and plasmid in insect cells pBacPAK8 / 9 and the like.
As the enhancer sequence and terminator sequence, those commonly used by those skilled in the art, such as those derived from SV40, respectively, can be used.
As the selection marker, a commonly used marker can be used by a conventional method. Examples include tetracycline, ampicillin, or antibiotic resistance genes such as kanamycin or neomycin, hygromycin or spectinomycin.
The expression vector can be prepared by linking at least the above-mentioned promoter, initiation codon, gene encoding the desired antibacterial protein, termination codon, and terminator region to appropriate replicable units in a continuous and circular manner. At this time, an appropriate DNA fragment (for example, a linker, other restriction enzyme sites, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase, if desired.
Introduction of the expression vector of the present invention into a host cell [transformation (transfection)] can be performed using a conventionally known method.
For example, in the case of bacteria (E. coil, Bacillus subtilis, etc.), the method of Cohen et al. [Proc. Natl. Acad. Sci, USA, 69, 2110 (1972)], protoplast method [Mol. Gen. Genet. 168, 111 (1979)] and the competent method [J. Mol. Biol. , 56, 209 (1971)], in the case of Saccharomyces cerevisiae, for example, the method of Hinnen et al. [Proc. Natl. Acad. Sci. USA, 75, 1927 (1978)] and lithium method [J. Bacteriol. , 153, 163 (1983)], in the case of plant cells, for example, by leaf disk method [Science, 227, 129 (1985)], electroporation method [Nature, 319, 791 (1986)]. For example, Graham's method [Virology, 52, 456 (1973)], and for insect cells, for example, the method of Summers et al. [Mol. Cell. Biol. 3, 2156-2165 (1983)].
For the purpose of producing a disease resistant plant using the DNA fragment of the present invention, a plant transformation vector is useful. The plant vector is not particularly limited as long as it has the ability to express the gene in plant cells and produce the protein. For example, pBI221, pBI121 (manufactured by Clontech), and these are derived from these. Vector. In particular, for transformation of monocotyledons, pIG121Hm, pTOK233 (above Hiei et al., Plant J., 6, 271-282 (1994)), pSB424 (Komari et al., Plant J., 10, 165-174 (1996). )) Etc.
The transformed plant can be prepared by constructing a plant transformation vector by replacing the DNA fragment of the present invention with the β-glucuronidase (GUS) gene site of the above-mentioned vector and introducing the vector into a plant. The plant transformation vector preferably contains at least a promoter, a translation initiation codon, a desired gene (a DNA sequence of the present invention or a part thereof), a translation termination codon, and a terminator. Further, it may appropriately contain a DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5 'side and 3' side of the desired gene, a selection marker region, and the like.
The promoter and terminator are not particularly limited as long as they function in plant cells. Examples of promoters for constitutive expression include the actin and ubiquitin gene promoters in addition to the 35S promoter previously incorporated in the vector. Is done. However, more preferably, an inducible promoter may be incorporated and used. In this way, the protein is produced and becomes resistant only when the pest contacts the transformed plant. Inducible promoters to be used include promoters of phenylalanine ammonia lyase, chitinase, glucanase, thionine, ozmotin or other genes that respond to pests and stress.
As a method for introducing a gene into a plant, a method using Agrobacterium (Horsch et al., Science, 227, 129 (1985), Hiei et al., Plant J., 6, 271-282 (1994)), electro Polation method (From mm et al., Nature, 319, 791 (1986)), PEG method (Paszkowski et al., EMBO J., 3, 2717 (1984)), microinjection method (Crossway et al., Mol. Gen, Genet., 202, 179 (1986)), micro-collision method (McCabe et al., Bio / Technology, 6, 923 (1988)) and the like. If it is a method, it will not specifically limit. In addition, the plant species serving as a host is not particularly limited as long as it is compatible with the plant transformation vector of the present invention and can be transformed, and in plants normally used in the technical field of the present invention, for example, dicotyledonous plants. Examples include tobacco, Arabidopsis, tomato, cucumber, carrot, soybean, potato, sugar beet, turnip, Chinese cabbage, rapeseed, cotton, petunia, and monocotyledonous plants such as rice, corn, and wheat.
The protein of the present invention has extremely strong antibacterial activity. For example, spore germination is completely suppressed at a very low concentration of 5 ng / ml against rice blast fungus (see Example 2 described later). At this concentration, spore germination is not observed even after prolonged incubation, and therefore the effect of the protein of the present invention on blast fungus is considered to be a bactericidal effect rather than a partial inhibition of growth. No antibacterial protein capable of completely inhibiting the growth of pathogenic bacteria at such a low concentration (nanogram order) has been reported to date as far as the present inventors know. In the examples described below, rice blast fungus and blight fungus, which are the most important diseases of rice, were used as plant pathogens for antibacterial assays for the purification of antibacterial proteins. The possibility of having an antibacterial effect at a level that is not much different from the plant disease of is very high. Thus, since the antibacterial protein derived from the hon-shimeji mushroom of the present invention has a strong antibacterial activity, the protein of the present invention can be used as a preparation that is contained in an active form as a drug such as an antibacterial agent or agricultural chemical. Can be used. In this case, if the DNA sequence encoding the protein of the present invention is used, it can be produced in large quantities by incorporating the DNA into an expression vector that functions in, for example, Escherichia coli or yeast as described above.
The protein of the present invention can be expressed (produced) by culturing transformed cells containing the expression vector prepared as described above in a nutrient medium. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol and the like. Examples of the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like. In addition, other nutrients (for example, inorganic salts (for example, sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)) are included as desired. You may go out. The culture is performed by a method known in the art. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the protein of the present invention is produced in large quantities. For example, in Example 4 of the present invention, the recombinant antibacterial protein of the present invention was expressed using Escherichia coli (M15) as a host cell. Although not limited thereto, in the case of expression in E. coli, the culture conditions for recombinant protein expression are preferably cultured at a temperature of 4 ° C. to 40 ° C. and induced by 0.01 mM to 5.0 mM IPTG. I do.
The protein of the present invention can be obtained from the culture obtained by the above culture as follows. That is, when the protein of the present invention accumulates in a host cell, the host cell is collected by an operation such as centrifugation or filtration, and this is collected into an appropriate buffer solution (for example, a Tris buffer solution having a concentration of about 10 M to 100 mM, phosphorous). Acid buffer, HEPES buffer, MES buffer, etc. A method suitable for the host cell to be used after suspending in a buffer whose pH varies depending on the buffer used, but preferably in the range of pH 5.0 to 9.0. The cells are disrupted by centrifugation and the contents of the host cells are obtained by centrifugation. On the other hand, when the protein of the present invention is secreted outside the host cell, the host cell and the medium are separated by an operation such as centrifugation or filtration to obtain a culture filtrate. The host cell disruption solution or culture filtrate can be used for purification or isolation of the protein of the present invention as it is or after dialysis and ammonium sulfate precipitation.
Examples of the purification / isolation method include the following methods. That is, when a tag such as 6 × histidine, GST, or maltose binding protein is attached to the protein, a method by affinity chromatography suitable for each tag generally used can be mentioned. For example, but not limited to, in Example 4 described later herein, a recombinant antibacterial protein having a 6 × histidine tag at the N-terminus was expressed. The recombinant protein was purified using Ni-NTA agarose (Qiagen) having affinity for 6 × histidine. On the other hand, when the protein of the present invention is produced without attaching such a tag, for example, a method described in detail in Examples described later, that is, a method by ion exchange chromatography can be mentioned. In addition to this, a method of combining gel filtration, hydrophobic chromatography, isoelectric point chromatography and the like can also be mentioned.
As described above, the antibacterial protein of the present invention can be used for the prevention and treatment of plant diseases involving fungi or bacteria. That is, according to the present invention, an antibacterial agent comprising the antibacterial protein of the present invention as an active ingredient is provided. The antibacterial agent of the present invention can usually be applied systemically or locally in plants.
Application amount is plant type, growth stage, symptom, application method, treatment time, type of protein to be applied (full-length protein, protein with substitution, deletion, insertion and / or addition of part of the protein, etc.) Depending on the climate of the growing place, the soil of the growing place, etc., it can be sprayed once to several times a day. The amount of application varies depending on various conditions. When spraying the antibacterial agent of the present invention, it is possible to mix and spray a solution, a suspension, an emulsion, and the like as necessary. As an aqueous or non-aqueous solution or suspension, one or more active substances are mixed as at least one inert diluent. Examples of the aqueous diluent include distilled water and saline. Examples of non-aqueous diluents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and alcohols such as ethanol.
Such antibacterial compositions may further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersing agents or stabilizers (eg arginine, aspartic acid, etc.).
These are sterilized by filtration through a bacteria-retaining filter, blending with a bactericide, or irradiation as necessary. These can also be produced in the form of a sterile solid composition by, for example, a freeze-drying method and dissolved in sterile distilled water or other solvent before use.
The dosage form of the antibacterial agent thus obtained may be determined according to the application to be used, mixed with the above additives, and in the form of tablets, pills, powders, granules, liquids, emulsions, etc. Can be sprayed.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
Example
Example 1 Construction of assay system
1) Establishment of a test system
Cultivation of pathogenic bacteria: Rice blast fungus (strain SUS-1, race 337; sold by Tohoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries) was cultured on automill medium (Difco, 1% sucrose added) to obtain conidia. . The spore solution was added at 10% glycerol and stored at -80 ° C.
Rice blight fungus (strain JT872) was cultured in 1/2 potato broth medium (PD medium: manufactured by Difco) for 2 days, and 3 mycelial masses of about 5 × 5 mm were mixed with 1/2 PD medium using Teflon homogenizer. Fragmented mycelia obtained by light grinding were used as inoculum.
Inoculate the above inoculum with a 96-well microtiter plate (manufactured by Corning) in a volume of 100 μl so that the number of conidia of blast fungus per well is about 1,000, and the number of fragmented hyphae is about 300. / 2PD medium was added and cultured in a 28 ° C incubator. The growth of the bacteria was measured by measuring the absorbance at 595 nm with a microplate reader (Benchmark manufactured by Bio-Rad).
Effect of salt and buffer: Determine the effect of salt and buffer on the growth of bacteria by adding a certain amount of NaCl, phosphate buffer, Tris buffer, Hepes buffer, bovine serum albumin, dithiothreitol, etc. to the medium did.
2) Protein extraction from Honshimeji
Honshimeji (produced in Japan; obtained from Shiga Prefectural Forest Center) was cut in advance with scissors, frozen in liquid nitrogen and ground to a fine powder in a mortar, and 30 ml of 50 mM Hepes buffer was added to add 30 Extraction was performed for minutes. The extract is filtered through Miracloth and 10,000_×g. Ammonium sulfate was added to the supernatant after centrifugation for 20 minutes to 75% saturation, and the mixture was allowed to stand at 4 ° C. overnight. Again, 15,000_×g. Precipitate by centrifugation for 20 minutes, dissolve the precipitate in 3 ml of 10 mM Hepes buffer, pH 7.5, and use a dialysis tube (Spectra / Por1 MWCO 6-8000, manufactured by Spectrum Medical Industries) or a benzoylated dialysis tube. Dialysis was performed against 10 mM Hepes buffer, pH 7.5 using (manufactured by SIGMA), and insoluble matters were removed by centrifugation to obtain a hon-shimeji protein sample. The protein concentration of the hon-shimeji protein sample was measured by the Bradford method using bovine serum albumin (BSA) as a standard protein.
Example 2 Purification of antibacterial protein
1) Antibacterial activity of hon-shimeji crude protein sample
Determine the presence or absence of antibacterial activity by adding a certain amount of hon-shimeji mushroom crude protein extract to the culture system of blast fungus and blight fungus immediately after the start of cultivation, and culturing for up to 2 days. did. For protein samples, a dilution series was prepared and the dilution limit for antibacterial activity was detected. As a result, high antibacterial activity against both pathogens of rice blast and crest was found (Table 1).

The complete growth-inhibiting concentration against blast fungus was estimated to be 30 μg total extracted protein / ml or less. From this, it was clarified that the hon-shimeji mushroom extract contains a substance having high antibacterial activity. As for the mode of growth inhibition of this protein extract against bacteria, complete suppression of germination was observed when the concentration was high, and inhibition of hyphal growth was observed when the concentration was low. The degree of inhibition of hyphal elongation was clearly concentration dependent. In addition, the cells of the blast fungus had a cytoplasm-like appearance with the cytoplasm separated from the cell wall.
2) Purification by ion exchange column
Next, the antibacterial protein was purified. Honshimeji (70 g) was pulverized in liquid nitrogen, and the protein was extracted in 200 ml of a buffer (50 mM MES, 50 mM NaCl, pH 6.0) for 30 minutes. After filtration through double Miracloth, the mixture was centrifuged at 15000 × g for 20 minutes to precipitate impurities. The supernatant was further filtered with filter paper to obtain a protein sample. About 200 ml of the protein sample was applied to a column (inner diameter 1.1 cm × 20 cm) packed with an ion exchanger Q-SepharoseFF (manufactured by Pharmacia). The flow rate was 2.5 ml / min, the basic buffer was 50 mM Mes pH 6.0, 50 mM NaCl, the elution buffer was 50 mM Mes pH 6.0, 1 M NaCl, and the gradient (50 mM to 1 M in NaCl) It took 120 minutes from 100 minutes after hitting. This was followed by a further flow of elution buffer for 40 minutes. Fractions were collected 4 times every 40 minutes from the start of the gradient (100 ml / fraction). A dilution series of these four fractions (I, II, III, IV) was prepared and an antibacterial assay for rice blast fungus was performed. As a result, antibacterial activity was observed in fractions II to IV. These fractions resulted in protoplast separation in the pathogenic cells. Fraction II (corresponding to 0 to 333 mM NaCl) having the highest activity was concentrated with Centriprep 10 (Amicon, MWCO 10,000), applied to an ion exchange column MonoQHR5 / 5 (Pharmacia), and the antibacterial protein was partially applied. Purified. The flow rate was 1 ml / min, the basic buffer was 50 mM Mes pH 6.0, 50 mM NaCl, the elution buffer was 50 mM Mes pH 6.0, 1 M NaCl, and the gradient (50 mM to 1 M NaCl) was sampled. It took 40 minutes after 20 minutes. A part of each fraction (1 ml) was subjected to an antibacterial assay for blast fungus and SDS-PAGE electrophoresis. First, the relationship between the HPLC chart and the antibacterial strength is shown in FIG. Antibacterial activity was obtained by taking 5, 1 and 0.2 μl from each MonoQ fraction and assaying for blast fungus, ++ (inhibiting to 0.2 μl), ++ (inhibiting to 1 μl), + (inhibiting to 5 μl), − ( 4 levels are displayed. As a result, when the protein was monitored by antibacterial activity against A280 and blast fungus, an antibacterial protein elution peak appeared at an ionic strength (NaCl concentration) of around 250 mM at pH 6.0. Thereafter, it was found that the antibacterial activity of the protein per unit eluate gradually decreased as the ionic strength increased.
Next, 10 μl was taken from each fraction, an equal volume of 2 × SDS electrophoresis buffer (Sambrook et al. 1989) was added, treated at 95 ° C. for 5 minutes, and then subjected to SDS-PAGE electrolysis according to the method of Laemmli (1970). Electrophoresis was performed. For the gel, 15% PAGEL (manufactured by ATTO) was used, and the protein was detected by silver staining II kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.). In order to estimate the approximate molecular weight and amount of protein, a molecular weight marker (LMW: manufactured by Pharmacia LKB, 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, 14.4 kDa) in order of molecular weight becomes 20 ng per band. Was run as follows. The relationship between the electrophoretic image and the antibacterial strength is shown in FIG. The numbers displayed above each lane are the same as the fraction numbers in FIG. 1, and the antibacterial strength is also displayed according to FIG. A close examination of the bands of proteins thought to be linked to antibacterial properties suggested two bands of about 70 kDa and about 65 kDa (arrows in FIG. 2). Since the concentration of these two bands and the degree of antibacterial activity are positively correlated, it was strongly suggested that these bands may be antibacterial protein bodies. Among them, the 65 kDa protein was very well linked to the concentration of the protein band and the strength of the antibacterial activity both in the fractionation by Q-Sepharose and the fractionation by MonoQ. The amount of antibacterial protein was estimated from the molecular weight marker (67 kDa albumin) with a densitometer, and the complete growth inhibitory concentration against blast fungus was calculated to be about 5 ng / ml.
3) Decoding the N-terminal amino acid sequence of antibacterial proteins
MonoQ fraction numbers 36 to 44 were concentrated with Centricut V-20 (Kurabo) and subjected to SDS-PAGE electrophoresis. Tris was removed, transferred to a PVDF membrane (manufactured by Millipore) using a buffer system not containing glycine, lightly stained with Coomassie Brilliant Blue R-250, and then decolorized. Thereafter, 70 kDa and 65 kDa protein bands considered to be antibacterial proteins were cut out. The N-terminal amino acid sequence was determined by Edman degradation method using a gas phase protein sequencer (HPG1005A Protein Sequencing System).
As a result, the following 30 amino acids from the 65 kDa protein:
N-terminal-NAEEGTAVVPYVPPGYHKKKNEIEFQKDIDRFV-C-terminal (SEQ ID NO: 3)
Was decided.
On the other hand, the 70 kDa protein could not be decoded. As this cause, it was considered that the N-terminal was blocked. Therefore, the 70 kDa protein was partially digested using lysyl endopeptidase and V8 protease to obtain a 43 kDa lysyl endopeptidase digest and a 45 kDa V8 protease digest. As a result of reanalyzing the amino acid sequences of these partially digested proteins, 24 residues from the former, N-terminal-EFDESIRHTLVRLSDLQDAYKDRQR-C-terminal (SEQ ID NO: 4)
29 residues from the latter
N-terminus-AERLIGTSTKEFDDESIRHTLVLRRQDAY-C-terminus (SEQ ID NO: 5)
Was decided. Since both amino acid sequences largely overlapped, the internal amino acid sequence obtained by partial degradation was 34 residues in total.
N-terminus-AERLIGTSTKEFDESIRHTLVLRRSQDAYKDRRQ-C-terminus (SEQ ID NO: 6)
Is decided. A database search was performed on the determined amino acid sequence. As a result, 30 amino acids from the 65 kDa protein and 34 amino acids from the 70 kDa protein both showed homology with the piranose oxidase of Kawaratake. Therefore, the pyranose oxidase activity of each fraction of MonoQ was measured. The measuring method was based on the method of Nishimura et al (1996). As a result, the high pyranose oxidase activity and the strength of antibacterial activity were in good agreement. Therefore, antibacterial activity was presumed to be derived from hydrogen peroxide produced when glucose in the medium was oxidized by the enzyme. Next, fractions containing only both the 65 kDa and 70 kDa proteins (FIG. 2, numbers 42 to 44) were concentrated, and various properties of pyranose oxidase were analyzed. As a result, it was clarified that honshimeji-derived antibacterial protein has a very high pyranose oxidase activity and a low Km value for glucose and 1,5-anhydroglucitol (Table 2).

* 1U = 1 μmole H₂O₂/ Min 37 ° C in pH 7.0
The amount of enzyme protein (65 kDa + 70 kDa) was quantified by SDS-PAGE silver staining.
Example 3 Isolation of cDNA
1) Degenerate primer design
Based on the amino acid sequence determined in 1), a primer in which all possible bases were mixed was synthesized (Tm is 52 to 56 ° C., and the number in parentheses indicates degeneracy). Specifically, the following three types of primers were synthesized from an amino acid sequence (30 residues) derived from a 65 kDa protein.
65R1 (5'-gargargia gicigticcc-3 '(4)) (SEQ ID NO: 7)
65R2 (5'-gartycaragayathaymg-3 '(384)) (SEQ ID NO: 8)
65R3 (5'-ttygiaaygthiattgyggigc-3 '(24)) (SEQ ID NO: 9)
On the other hand, the following three types of primers were synthesized from an amino acid sequence (34 residues) derived from a partially decomposed product of 70 kDa protein.
70F1 (5'-tickickdataytcrtcraytc-3 '(384)) (SEQ ID NO: 10)
70F2 (5'-tgicktcyttrttaiigcrtcytg-3 '(64)) (SEQ ID NO: 11)
70F3 (5'-gigcraadicticgickrtc-3 '(96)) (SEQ ID NO: 12)
In the above primers, r is a or g, y is c or t, h is a or c or t, m is a or c, k is g or t, d is a or g or t, s represents g or c, w represents a or t, and i represents inosine.
2) Construction of Honshimeji fruit body cDNA library
Total nucleic acid was extracted from Honshimeji fruiting body by SDS phenol method, and total RNA was recovered by lithium chloride precipitation. From this, hon-shimeji mRNA was prepared using an mRNA purification kit (Pharmacia). About 10 to 20 μg of mRNA was obtained from fruiting bodies. Of these, 5 μg was used in a ZAP cDNA synthesis kit (Stratagene) to synthesize cDNA. A 1-5 kb cDNA was fractionated with a gel filtration column, ligated to a Uni-ZAP XR vector (Stratagene), and packaged with Gigapack III (Stratagene). All procedures followed the instructions included with the kit. The titer of the constructed Hongshimeji cDNA library was calculated to be about 3 million pfu.
3) Acquisition of probe by RT-PCR
Using the primers synthesized in 1), PCR was performed using the cDNA synthesized in 2) as a template, and an attempt was made to amplify a partial length cDNA of hon-shimeji protein, which serves as a probe for screening the library. The reaction conditions were as follows. In a 50 μl reaction, 100 ng of cDNA, 10_×Ex taq buffer 5 μl, dNTPs 4 μl, primer 10 pmoles / each kind of sequence, Ex taq (manufactured by Takara) + Taq START antibody (manufactured by Clontech) 700 μm, TE 94 ° C for 3 minutes, 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute 35 times, and 72 ° C for 6 minutes once. As a result, a product of about 0.4 to 0.5 kb was amplified in the primer combinations of 65R1-70F1, 65R1-70F2, 65R2-70F1, 65R2-70F2, 65R2-70F3, and 65R3-70F1. Among these, a fragment of about 0.4 kb by the combination of 65R2-70F1 having higher amplification efficiency was gel purified, cloned into the vector pCRII (manufactured by Invitrogen), and the nucleotide sequence was determined. The amino acid sequence deduced from this base sequence contained a sequence identical to a part of the amino acid sequence determined in 23), and showed a loose homology with the oyster mushroom pyranose oxidase over the entire sequence. From this, it was confirmed that the purified 70 kDa and 65 kDa antibacterial proteins were encoded by one gene, and the cDNA clone obtained by RT-PCR was a partial length cDNA of Honshimeji antibacterial protein.
4) Screening of full-length cDNA
The clone obtained in 3) was excised from the vector, and this was used as a probe to screen the Hongshimeji cDNA library constructed in 2). According to the instructions of ZAP cDNA synthesis kit (manufactured by Stratagene) on a 14 × 10 cm square petri dish, about 15000 pfu of phage was plated together with the host fungus XL1-blue MRF '. Plaques were brought into contact with Hybond-N + nylon membrane filter (Amersham) and subjected to alkali treatment according to the instructions attached to the membrane to denature the DNA and fix it on the membrane. Hybridization and washing conditions were performed under high stringency according to the instructions attached to the membrane. In the primary screening, 20 positive clones were obtained from about 120,000 pfu of phage. These clones were subjected to secondary screening and tertiary screening, which was also plaque purification, and in vivo excision was performed on all 20 clones according to the description of ZAP cDNA synthesis kit (Stratagene). As a result, 18 clones were recovered as cDNA incorporated into the phagemid vector pBluescript SK. These clones were 1.7-2.1 kb in length, suggesting that they were derived from very similar genes from restriction enzyme analysis.
5) Determination of base sequence
About the 18 cDNA clones described above, approximately 500 bp of 5 'and 3' base sequences were determined. The obtained base sequence data was transferred to Genetyx ver. Analysis was performed using 9.0 analysis software (manufactured by Software Development). As a result, although the poly A addition site was different between clones, all clones contained a DNA sequence encoding 30 amino acids of the 65 kDa protein determined in 23). Since the cDNA clone of No. 13 (2.1 kb) was the longest, the entire nucleotide sequence of this clone was determined. The nucleotide sequence was determined by the primer walking method using an ABI PRISM fluorescence sequencer (Mode 1310 Genetic Analyzer, manufactured by Perkin Elmer). As a result, the cDNA encoding Honshimeji antibacterial protein consisted of a total length of 2106 base pairs, contained an open reading frame of 1854 base pairs, and encoded 618 amino acids (SEQ ID NOs: 1 and 2). The molecular weight estimated from the amino acid sequence was calculated to be about 68487, and the isoelectric point was 6.12. In the amino acid sequence determined from the purified protein, 30 amino acids derived from 65 kDa corresponded to amino acids 76 to 105 in SEQ ID NO: 2 in the sequence listing, and 34 amino acids derived from 70 kDa corresponded to 211 to 244 in the same. This means that the 65 kDa and 70 kDa proteins are encoded by the same gene. Furthermore, there were seven putative glycosylation sites (amino acid numbers 154, 319, 360, 412, 558, 573, 583 in SEQ ID NO: 2 in the sequence listing).
From the above results, it was concluded that the cloned cDNA was derived from a gene encoding hon-shimeji antibacterial protein. When the homology search (BLAST) was performed on the database (DDBJ) for the amino acid sequence of the hon-shimeji mushroom-derived antibacterial protein of the present invention, it had 45% identity as a whole with the amino acid sequence of Kawaratake pyranose oxidase. Since there is no significant homologous sequence other than this, this gene is considered to be a gene encoding a novel pyranose oxidase-like protein.
Example 4 Expression in E. coli and purification of recombinant protein
1) Construction of expression vector
The cDNA clone No. 13 isolated in Example 3 5) has a unique EcoT22I restriction site about 0.06 kb downstream of the translation stop codon and a unique BamHI restriction about 0.25 kb upstream of the translation stop codon. The site exists. BamHI exists at the multiple cloning site on the 5'-side vector of this cDNA. Therefore, the plasmid (vector is pBluescript) in which this cDNA was incorporated was first completely digested with the restriction enzyme EcoT22I (Takara) and then partially digested with BamHI (Takara). The resulting approximately 2 kb BamHI (BamHI on the vector) -EcoT22I fragment was incorporated into an expression vector pQE30 (manufactured by Qiagen) double-digested in advance with restriction enzymes BamHI and PstI (named pQEHSPOfull). The completed construct contains the longest open reading frame (ORF) of the full-length cDNA encoding the hon-shimedipyranose oxidase-like protein of the present invention (the 8th to 1864th base sequences of SEQ ID NO: 1 in the sequence listing). This encodes the entire amino acid sequence of SEQ ID NO: 2 in the sequence listing), and 6 histidine residues are added as tags to the N-terminus of the expressed protein.
2) Expression in E. coli
Expression experiments were performed using the host E. coli strain M15. The bacterial culture method and the protein expression induction method by IPTG (Isopropyl β-D-Thiogalactopyranoside) were in accordance with the instructions of Qiagen. OD in LB medium containing antibiotics ampicillin and kanamycin₆₀₀Bacteria were pre-cultured until about 0.5, and then cultured in the same medium at various temperatures and various IPTG concentrations for a certain period of time to induce expression. Soluble protein was extracted from the cells according to the instructions of Qiagen, and a certain amount thereof was analyzed by Nishimura et al. (1996), pyranose oxidase activity was measured. The results of recombinant protein expression are summarized in Table 3.

First, induction was attempted under normal induction conditions of a culture temperature of 37 ° C. and an IPTG concentration of 2 mM. Although insoluble inclusion bodies were expressed in large quantities, the pyranose oxidase activity of the soluble fraction was not detected. Therefore, expression was induced under various conditions, and the pyranose oxidase activity of the soluble fraction was measured. As a result, the pyranose oxidase activity increased as the induction conditions were relaxed, that is, as the culture temperature and the IPTG concentration were lowered. This was thought to be due to the increase in soluble recombinant protein content due to relaxation of the induction conditions. On the other hand, no activity was detected in the control pQE30 (vector only). From the above results, it has been clarified that the cloned cDNA encodes an active pyranose oxidase-like protein. In addition, the culture | cultivation in 21 hours compared with the culture | cultivation for 5 hours at the culture temperature of 25 degreeC and IPTG density | concentration of 0.5 mM reduced the activity. This probably suggests that the expressed protein has been degraded.
Next, purification of the expressed protein was attempted. Recombinant pyranose oxidase-like protein was purified using Ni-NTA agarose (manufactured by Qiagen) from a soluble protein fraction derived from cells whose expression was induced at a culture temperature of 16 ° C. and an IPTG concentration of 0.1 mM. When protein was adsorbed, washed and eluted according to the instructions of Qiagen, pyranose oxidase activity was detected only in the eluted fraction. Therefore, the N-terminus of the recombinant protein is not degraded in E. coli, and the histidine residue is bound to the N-terminus, and the recombinant protein can be easily purified by Ni-NTA agarose using this. In addition, it was revealed that the entire coding region of the cloned full-length cDNA encodes an active protein as it is (without removing the portion corresponding to the N-terminal side of the protein). The yield of recombinant protein was estimated to be several mg per 11 E. coli culture.
effect
If a preparation comprising a protein component characterized by including the sequence of SEQ ID NO: 2 in the sequence listing of the present invention or the entire sequence excluding the first to 75th sequences among them is produced, Expected to be used as an antibacterial agent. Moreover, if a reagent containing the protein component as an active ingredient is prepared, it can be used for measurement of sugars such as blood sugar. Of the DNA sequence of SEQ ID NO: 1 in the sequence listing of the present invention, it is a DNA sequence characterized by the 8th to 1864th sequence, or a 233rd to 1864th sequence, The DNA sequence to be incorporated into an expression cassette of an appropriate constitutive, organ / time-specific or stress or pest-inducible promoter sequence that can function in plant cells, and a terminator sequence that can function in plant cells, It is expected that pest-resistant plants can be produced by introduction into plant cells and obtaining regenerated individuals. Alternatively, the protein can be obtained in a large amount at a low cost by introducing and expressing the above DNA sequence into E. coli, yeast, insects or certain animal cells using expression vectors that can be amplified in each host. it can.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the relationship between the anti-bacterial activity and the chart of the separation of hon-shimeji protein by the MonoQ column.
FIG. 2 shows the relationship between the electrophoretic image and antibacterial properties of the separation of hon-shimeji antibacterial protein by the MonoQ column. The numbers on the lanes correspond to the fraction numbers in FIG. 1, and M indicates a molecular weight marker. Symbols (−, +, ++, +++) below the lane indicate the strength of antibacterial activity. Antibacterial proteins (70 kDa and 65 kDa) are indicated by arrows.

Claims (17)

An antibacterial protein having an antibacterial activity against rice blight fungus or rice blast fungus, having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing or an amino acid sequence having 90% or more homology with this sequence . The antibacterial protein according to claim 1 , which has an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. A polypeptide comprising the partial amino acid sequence 76-618 of SEQ ID NO: 2 in the sequence listing, and a polypeptide having an amino acid sequence having 90% or more homology with the above amino acid sequence , which is against rice blight fungus or rice blast fungus An antibacterial protein comprising the polypeptide exhibiting antibacterial activity, alone or in combination of any of the polypeptides. Recovering a fraction precipitated by a sulfuric acid precipitation method using 75% saturation of ammonium sulfate from an aqueous extract of hon-shimeji; and subjecting the fraction to ion exchange chromatography to elute at a NaCl concentration of 0.05M to 1M. Recovering
The manufacturing method of the antibacterial protein of Claim 1 or 3 containing this. A gene encoding the antibacterial protein according to claim 1 or 3 . The gene according to claim 5 , which has the base sequence set forth in SEQ ID NO: 1 in the sequence listing or a base sequence that hybridizes with the base sequence under stringent conditions. The gene according to claim 5 , which has a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. The gene according to claim 5 , which has a base sequence having 95% or more homology with the base sequence shown in SEQ ID NO: 1 in the sequence listing. A pair of oligonucleotides for obtaining a gene encoding an antibacterial protein derived from Honshimeji,
Two regions are selected from the base sequence of the gene encoding the antibacterial protein shown in SEQ ID NO: 1 in the sequence listing so as to satisfy the following conditions:
1) The length of each region is 15-30 bases;
2) The percentage of G + C in each region is 40-60%;
A single-stranded DNA having the same base sequence as the above region or a base sequence complementary to the above region is produced, or the genetic code is degenerate so as not to change the amino acid residue encoded by the single-stranded DNA. A pair of said oligonucleotides produced by a method comprising producing a mixture of single stranded DNAs under consideration. The method according to claim 9, comprising one oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 7 to 9 in the sequence listing, and one oligonucleotide having a nucleotide sequence selected from SEQ ID NOs: 10 to 12. The described oligonucleotide pair . Using the pair of oligonucleotides according to claim 9, a nucleic acid amplification reaction is carried out using a hon-shimeji fruit body cDNA library as a template, and a part of the gene encoding the antibacterial protein according to claim 1 is amplified. A method for isolating a gene according to claim 5 , comprising screening the cDNA library using the amplified product as a probe to isolate a full-length cDNA clone. A recombinant vector comprising the gene according to claim 5 . The recombinant vector according to claim 12 , wherein the vector is an expression vector. A transformant obtained by introducing the recombinant vector according to claim 12 into a host organism. The method for producing an antibacterial protein according to claim 1 or 3 , comprising culturing the transformant of claim 1 under conditions that promote expression of the antibacterial protein. A recombinant antibacterial protein obtained by the method according to claim 15 . An antibacterial agent comprising the antibacterial protein according to claim 1 or 3 as an active ingredient.

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